In some cases a desired insertion of a recombinant transcription unit into an organism may be limited by patent rights, e.g. directed to vectors comprising the transcription unit or methods using the transcription unit. Some of such limitations may be avoided by in vivo assembly of transcription units, e.g. in the field of gene suppression.
Gene suppression may be effective against a native gene associated with a trait in a plant or animal, e.g. to provide reduced levels of a protein encoded by the native gene and might lead to altered levels of the corresponding substrate and metabolites. Gene suppression can also be effective against target genes in pest or pathogen organisms, e.g. a virus, insect or nematodes that may feed on or infect an organism containing gene suppression agents. Gene suppression includes any of the well-known methods for suppressing transcription of a gene or the accumulation of the MRNA corresponding to that gene, thereby preventing translation of the transcript into protein.
More particularly, gene suppression mediated by inserting a recombinant DNA construct with anti-sense oriented DNA to regulate gene expression in plant cells is disclosed in U.S. Pat. No. 5,107,065 (Shewmaker et al.) and U.S. Pat. No. 5,759,829 (Shewmaker et al.). Plants transformed using such anti-sense oriented DNA constructs for gene suppression can comprise integrated DNA arranged as an inverted repeat that resulted from co-insertion of several copies of the transfer DNA (T-DNA) into plants by Agrobacterium-mediated transformation, as disclosed by Redenbaugh et al. in “Safety Assessment of Genetically Engineered Flavr Savr™ Tomato, CRC Press, Inc. (1992). Inverted repeat insertions can comprise a part or all of the T-DNA, e.g. contain an inverted repeat of a complete or partial anti-sense construct. Screening for inserted DNA comprising inverted repeat elements can improve the efficiency of identifying transformation events effective for gene silencing when the transformation construct is a simple anti-sense DNA construct.
Gene suppression triggered by inserting a recombinant DNA construct with sense-oriented DNA to regulate gene expression in plants is disclosed in U.S. Pat. No. 5,283,184 (Jorgensen et al.) and U.S. Pat. No. 5,231,020 (Jorgensen et al.). Inserted T-DNA providing gene suppression in plants transformed with such sense constructs by Agrobacterium is organized predominately in inverted repeat structures, as disclosed by Jorgensen et al., Mol. Gen. Genet., 207: 471-477 (1987). See also Stam et al., The Plant Journal, 12: 63-82 (1997) and De Buck et al., Plant Mol. Biol. 46 433-445 (2001), who used segregation studies to support Jorgensen's finding that in many events gene silencing is mediated by multimeric transgene T-DNA where the T-DNAs are arranged in inverted repeats. Screening for inserted DNA comprising inverted repeat elements can improve the gene silencing efficiency when transforming with simple sense-orientated DNA constructs.
Gene silencing can also be effected by transcribing RNA from both a sense and an anti-sense oriented DNA using two separate transcription units, e.g. as disclosed by Shewmaker et al. in U.S. Pat. No. 5,107,065 where in Example 1 a binary vector was prepared with both sense and anti-sense aroA genes. Similar constructs are disclosed in International Publication No. WO 99/53050 (Waterhouse et al.). See also U.S. Pat. No. 6,326,193 where gene targeted DNA is operably linked to opposing promoters.
Gene suppression can be achieved in plants by providing transformation constructs that are capable of generating an RNA that can form double-stranded RNA along at least part of its length. Gene suppression in plants is disclosed in EP 0426195 A1 (Goldbach et al.) where recombinant DNA constructs for transcription into hairpin RNA provided transgenic plants with resistance to tobacco spotted wilt virus. See also Sijen et aL, The Plant Cell, Vol. 8, 2277-2294 (1996) which discloses the use of constructs carrying inverted repeats (sense followed by anti-sense) of a cowpea mosaic virus gene in transgenic plants to mediate virus resistance. See also International Publication No. 98/53083 (Grierson et al.) and related U.S. patent application Publication No. 2003/0175965 A1 (Lowe et al.) which disclose gene suppression, using a double stranded RNA construct comprising a gene coding sequence preceded by an inverted repeat of 5′UTR. Constructs for posttranscriptional gene suppression in plants by double-stranded RNA of the target gene are also disclosed in International Publications WO 99/53050 (Waterhouse et al.) and WO 99/49029 (Graham et al.). See also U.S. patent application Publication No. US 2002/0048814 A1 (Oeller) where DNA constructs are transcribed to sense or anti-sense RNA with a hairpin-forming poly(T)-poly(A) tail. See also U.S. patent application Publication No. US 2003/0018993 A1 (Gutterson et al.) where sense or anti-sense DNA is followed by an inverted repeat of the 3′ untranslated region of the NOS gene. See also U.S. patent application Publication No. US 2003/0036197 A1 (Glassman et al.) where RNA for reducing the expression of target MRNA comprises a part with homology to target mRNA and a part with complementary RNA regions that are unrelated to endogenous RNA.
The production of dsRNA in plants to inhibit gene expression, e.g. in a nematode feeding on the plant, is disclosed U.S. Pat. No. 6,506,559 (Fire et aL.). Multi-gene suppression vectors for use in plants are disclosed in U.S. patent application Ser. No. 10/465,800 (Fillatti).
All of the above-described patents, applications and international publications disclosing materials and methods for gene suppression in plants using recombinant DNA constructs are incorporated herein by reference.
Transcriptional suppression such as promoter trans suppression can be effected by expressing a DNA construct comprising a promoter operably linked to inverted repeats of promoter DNA from a target gene as disclosed by Mette et al., The EMBO Journal, Vol. 18, pp. 241-148, (1999) and by Mette et al., The EMBO Journal, Vol. 19, pp. 5194-5201-148, (2000), both of which are incorporated herein by reference.
Known methods for in vivo modification of recombinant DNA include homologous recombination and site-specific recombination. For instance, U.S. Pat. No. 6,750,379 discloses homologous recombination-mediated transgene alterations in plants, e.g. for marker deletion. Site-specific recombination of DNA in plant cells is disclosed in U.S. Pat. Nos. 5,658,772 and International Publications WO 02/088369 A1 and WO 2004/067749 A1. See also WO 2004/067748 A1 which discloses post transcription assembly by splicing of an artificial intron from transcribed RNA.
Co-integration studies of T-DNA are discussed by Krizkova et al. in Plant J., 16, 673-680 (1998) which reports assembling of a transcription unit from two copies of a T-DNA which has a promoterless gene at the 3′ end and an unlinked promoter at the 5′ end.